1. Field of the Invention
The invention relates to enzymes involved in the metabolism of cholesterol and more specifically to the cholesterol esterase secreted by the pancreas in mammals. Cholesterol metabolism is of critical interest to those involved in protecting human health. Atherosclerosis is the leading cause of death in the United States and reduction of serum cholesterol levels has recently been embraced as a national health priority. See NIH Consensus Panel Report, J.A.M.A. 253: 2094 (1985). NIH recommendations include measurement of serum cholesterol in all adults, with efforts to reduce cholesterol in those individuals with levels above 200 mg %. In this regard front line therapy is a reduction in the amount of cholesterol and triglycerides ingested, followed by the use of agents that interfere with absorption of ingested lipids. See Consensus Full Report, Arch. Inst. Med. 148: 36 (1988).
Pancreatic cholesterol esterase plays a pivotal role in the absorption of cholesterol and fatty acids. The inhibition of cholesterol esterase could lead to reduced serum cholesterol levels. Numerous approaches to developing inhibitors of cholesterol esterase will likely be attempted, including the use of chemical inhibitors. Therapeutic biologicals, such as monoclonal or polyclonal antibodies to pancreatic cholesterol esterase have great potential. In particular, antibodies against purified cholesterol esterase can be isolated from the milk of immunized cows and used as an ingestible therapeutic. Analogs of pancreatic cholesterol esterase are proteins similar to cholesterol esterase, but with sufficient variation in amino acid sequence to bind cholesterol esters without releasing free cholesterol and fatty acids. If such analogs can be developed they will serve as powerful inhibitors of cholesterol esterase function.
Whatever type of inhibitor is being developed, large quantities of highly purified enzyme are required to test the efficacy of any potential inhibitor, as well as to better understand the enzyme and thus allow the development of further therapeutic means. There is, therefore, a need for methods to purify useful quantities of homogeneous pancreatic cholesterol esterase. In addition, for the preparation of analog inhibitors, the amino acid sequence of the enzyme and its underlying DNA sequence must be known. Thus, there is a need for a cloned DNA sequence encoding cholesterol esterase, from which the DNA and amino acid sequences may be determined.
Finally, pancreatic cholesterol esterase has considerable commercial utility for enzymatic hydrolysis or synthesis of ester linkages in the preparation of biologicals or foodstuffs such as dairy products. There is, therefore, a need for a means of producing commercially significant, large-scale quantities of homogeneous cholesterol esterase, especially from cows, which cannot be met by purification of the enzyme from natural sources. What is needed, then, is a means for producing pancreatic cholesterol esterase through the use of recombinant DNA expression vectors in a suitable host cell or organism, as well as a means of large-scale purification of the enzyme so expressed.
2. Information Disclosure
Borja et al., 1964, Proc. J. Exp. Biol. and Med. 116: 496, teach that cholesterol esterase is secreted by the pancreas, and that its catalysis of cholesterol ester hydrolysis to produce free cholesterol and free fatty acids is essential for the absorption of cholesterol. Bosner et al., 1988, Proc. Natl. Acad. Sci. USA 85: 7438, teach that cholesterol esterase performs its function while anchored to the intestinal membrane via a receptor-like interaction with brush border membrane associated heparin. Lange and Spilburg, in co-pending application U.S. Ser. No. 340,868, teach sulfated polysaccharide inhibitors of human pancreatic cholesterol esterase which are effective in blocking the absorption of cholesterol and fatty acids into intestinal cells.
Numerous procedures for the preparation of pancreatic cholesterol esterase have been reported See, e.g., Allain et al., 1974, Clin. Chem. 20: 470, Calame et al., 1975, Arch. Biochem. Byophys. 168: 57, Labow et al., 1983, Biochem. Byophys. Acta 749: 32. In general, the reported procedures are tedious and give poor yields of heterogeneous material. Production of significant quantities of homogeneous material has not been achieved. For example, the commercially available cholesterol esterase, prepared by the method of Allain et al., is less than 5% pure according to both physical and functional assays. None of the existing preparative procedures has been useful for purifying cholesterol esterase from several different mammalian species.